The project's long term goal is to determine the molecular events that regulate neutrophil migration across the vascular endothelium. While neutrophil adhesion to the endothelium involves at least four classes of adhesion molecules, precisely how adhesion molecules regulate neutrophil migration is unknown. In vitro, greater than 75 percent of neutrophil migration occurs at tricellular corners where the borders of three endothelial cells converge and tight junctions are discontinuous. The two Specific Aims in this proposal are motivated by the working hypothesis that neutrophil migration at these sites is determined by the numbers, types, and spatial distribution of adhesion molecules and chemotactic factors on the endothelial surface. We will use genetically altered mice with targeted deletions of adhesion molecules in two areas of investigation. The first aim will determine in vitro the relative contribution of adhesion mechanisms to neutrophil migration at endothelial tricellular corners using mouse and human models. Specifically, isolated neutrophils and cultured endothelial cells will be used in adhesion assays to examine the functional role of leukocyte (CD11a/CD18 and CD11b/CD18) and endothelial (CD31, CD54, CD62E, CD62P) adhesion molecules and chemotactic factors (IL-8, PAF, MIP-2, and KC) in neutrophil migration at tricellular corners. For comparative purposes, the role of adhesion molecules in neutrophil migration across HUVEC monolayers will be assessed using blocking monoclonal antibodies. The spatial distribution of adhesion molecules and chemotactic factors will be characterized using immunogold scanning electron microscopy. The organization of tight junctions and the three-dimensional pathway taken by the neutrophil as it penetrates the endothelium will be determined by freeze-fracture transmission electron microscopy. The second aim will examine an in vivo model of acute inflammation in the mouse to determine if tricellular corners are preferred migration sites and if they are regulated by specific adhesion mechanisms. Specifically, TNFalpha, formyl peptide, or MIP-2 will be injected intrascrotally to induce neutrophil migration from the cremasteric microvasculature. The role of specific adhesion molecules in defining the migratory pathway will be assessed by light microscopy using silver stained whole mount preparations and by serial section reconstruction using transmission electron microscopy. Whether neutrophil migration affects endothelial tight junction organization will be assessed using freeze-fracture electron microscopy. Collectively, these studies will provide new insights into the molecular mechanisms regulating neutrophil migration across the endothelium. While leukocyte emigration is critical for host defense and normal healing, it is also partly responsible for a number of pathologic disorders (e.g., ischemia-reperfusion injury in the heart, rheumatoid arthritis, asthma, and emphysema). The proposed research will provide new data and insights for the design of anti-inflammatory therapies that target the multistep process of leukocyte migration.